1. Technical Field
Brassinosteroids are a class of plant growth regulatory substances found in low concentrations in all species of plants that have been examined. Brassinolide, one of the major types of the 40 or so brassinosteroids that have been described, was first discovered in the pollen of Brassica napus, (rapeseed), Grove et al., Nature 281:216, 1979.
Brassinosteroids produce a variety of useful phenotypic changes in plants that include: acceleration of seed germination and increased vigour of seedlings, increased cell size and elongation resulting in larger plants with increased biomass and seed yield, increased photosynthetic capacity and resistance to fungal and bacterial pathogens, Mandava et al., An. Rev. Plant Physiol. Plant Mol. Biol. 39:23, 1988.
The biosynthesis of sterols in plants begins with acetyl CoA and through sequential condensation reactions of the well characterized isoprenoid pathway leads to the formation of squalene, the immediate precursor of sterols in plants.
Further specific aspects of the biosynthesis of brassinosteroids have been discovered using cultured cells and brassinosteroid deficient mutations of Catharanthus, (Clouse et al., 1998; Fujoika et al., 2000). Plants with such mutations show a dwarf phenotype that may be rescued by the application of brassinosteroids demonstrating the involvement in one of the biosynthetic steps. Enzymes that are involved in the biosynthetic pathway include sterol desaturases (DWF7/STE1) (Choe et al., 1999b), oxidases (DWF1/DIM1/LKB) (Choe et al., 1999a; Nomura et al., 1999), reductases (DET2/LK) (Li et al., 1996), and cytochrome P450 hydroxylases (DWF4, CPD/DWF3, D) (Choe et al., 1998; Bishop et al., 1999). The known biochemical pathways resulting in the formation of brassinolide are reviewed in Noguchi et al., 2000.
In addition to enzymes involved in biosynthesis additional mutations in Arabidopsis and pea have revealed blockages in either brassinosteroid perception or transduction of brassinosteroid mediated responses. The BRI1 protein is a leucine-rich repeat serine/threonine kinase comprising a transmembrane spanning region and an external brassinosteroid binding domain believed to be a receptor protein required for brassinosteroid signal transduction across the plasma membrane. (Li and Chory, 1997; Wang, et al., 2001).
It is clear that brassinosteroids represent an important group of plant growth regulatory substances that affect many aspects of plant metabolism and growth and that modulation of the response to these substances can effect changes in plant development and performance of significant agronomic importance including seedling vigour, seed and biomass yield, tolerance to salt, heat, drought and disease and seed dormancy.
2. State of the Art
Brassinosteroids
Naturally occurring brassinosteroids are only found in minute concentrations in plants thus isolation and use of such compounds for application to plants to effect increases in growth and development are impractical because of the very high costs of extraction. Thompson et al., U.S. Pat. No. 4,346,226 describe the chemical synthesis of synthetic polyhydroxylated steroidal lactones that can be used as synthetic analogues of naturally occurring brassinolides.
Brassinosteroids are produced naturally as the result of a complex multi-step biosynthetic pathway. A number of transgenic approaches to manipulate one or more of the metabolic steps of brassinolide synthesis, degradation or perception have been described. Neff and Chory, U.S. Pat. No. 6,534,313 describe a gene, bas 1 and encoded protein BAS-1 that is a member of the cytochrome P450 family that appears to be a C-26 hydroxylase of brassinolide. Transgenic plants overexpressing the bas 1 gene are dwarfed and show depressed levels of brassinolide and some brassinosteroid precursors suggesting the role of this enzyme is inactivation of the growth promoting effects of brassinolide.
Koncz et al., U.S. Pat. No. 5,952,545 describe an additional P450 cytochrome hydrolylase gene and encoded protein involved in the early steps of brassinolide biosynthesis specifically in the conversion of cathasterone to teasterone. Plants with a mutated gene or a gene with reduced expression show a dwarf phenotype that is restored to wild type by brassinolide. Over expression of this gene however, does not produce plants with an increased growth or development phenotype seen with application of exogenous brassinolide.
Kang and Park, U.S. Pat. No. 6,605,469 describe a dark-inducible cytochrome P450 C-2 hydroxylase involved in the brassinosteroid biosynthetic pathway specifically through conversion of typhasterol to castasterone and 6-deoxotyphasterol to 6-deoxocasterone. The gene, designated ddwf1 and the encoded DDWF1 protein appear to be exclusively involved in the light/dark regulated development of hypocotyls. The DDWF1 protein was shown to interact with Pra-2 a small molecular weight GTP binding protein with known involvement in transduction of dark induced hypocotyl elongation. Transgenic plants overexpressing DDWF1 show increased elongation of hypocotyls in both light and dark, however other plants parts are not affected.
Chory and Wang, U.S. Pat. No. 6,768,043 describe an additional P450 cytochrome protein gene DAS5 involved in the biosynthesis of brassinosteroids. When the DAS5 gene is overexpressed in plants, higher levels of several brassinolide precursor molecules are recorded and transgenic plants exhibit an increased growth of up to 26% in fresh weight. The results achieved are consistent with an involvement of the DAS5 gene in the brassinosteroid pathway close to the formation of the active growth regulator brassinolide. Physiological effects of the overexpression of DAS5 in addition to increased growth were not reported.
Chory and Li, U.S. Pat. No. 6,245,969; U.S. Pat. No. 6,765,085 describe a trans-membrane receptor kinase gene, BIN 1 and transcribed polypeptide, comprising a brassinosteroid receptor. The BIN1 polypeptide has an extracellular brassinosteroid binding domain of 70 amino acids that functions as an extracellular domain receptor. The 70 amino acid island region is required for brassinosteroid binding whereas the trans-membrane receptor kinase activity transduces steroid signals across the plasma membrane. Overexpression of BIN1 in transgenic plants is suggested to result in plants characterized as having increased plant yield or vegetative biomass, however, examples of such effects are not provided.
Finally, Chory and Wang, U.S. Pat. No. 6,921,848, describe the BZR1 gene, and an altered bzr-1D gene involved in the brassinosteroid response pathway. The BZR1 protein and the bzr1-D protein appear to act downstream of the brassinosteroid receptor BIN 1. Expression of BZR1 genes in transgenic plants is proposed to cause such plants to grow larger and have increased yield in comparison with wild type plants however, examples of such effects are not provided.